Not applicable.
1. Field of the Invention
The present invention relates to a novel human gene encoding a polypeptide which is a member of the Kunitz-type protease inhibitor family. More specifically. isolated nucleic acid molecules are provided encoding a human polypeptide named Tissue Factor Pathway Inhibitor-3 hereinafter referred to TFPI-3. TFPI-3 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. Also provided are diagnostic methods for detecting disorders related to vascular hemostatsis and therapeutic methods for treating such disorders. The invention further relates to screening methods for identifying agonists and antagonists of TFPI-3.
2. Background Art
Proteases are responsible, either directly or indirectly, for all bodily functions, including cell growth, differentiation and death (apoptosis), cell nutrition, intra- and extracellular protein turnover (housekeeping and repair), cell migration and invasion. and fertilization and implantation. These functions extend from the cellular level to the organ and organism level to produce cascade systems such as hemostatis and inflamation, and complex processes at all levels of physiology and pathophysiology.
Maintenance of vascular integrity is an important host response to injury. Complex hemostatis mechanisms of coagulation, platelet function, and fibrinolysis exist to minimize adverse consequences of vascular injury and to accelerate vascular repair. Many of these hemostatic mechanisms are initiated and/or regulated by cells of the wall of the blood vessel.
Tissue-factor-pathway inhibitor (TFPI) is a cell-surface associated glycoprotein which plays a key role in the regulation of tissue factor-initiated blood coagulation. Human TFPI is a trace 42-kDa plasma glycoprotein that is synthesized primarily by endothelial cells and consists of a negatively charged amino terminal region, three tandem Kunitz-type inhibitor domains, and a highly basic carboxyl-terminal tail (Wun, T. C., et al., J. Biol. Chem. 263:6001 (1988)). After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines. TFPI forms a complex with factor Xa and inhibits its amidolytic and proteolytic activity. The factor Xa-TFPI complex rapidly inhibits activity of the factor VIIa-tissue factor complex.
The cloning and characterization of a gene coding for a second tissue factor pathway inhibitor (TFPI-2), has been reported (Sprecher, C. A. et al., Proc. Natl. Acad, Sci. USA, 91, 3353-3357 (1994)). This gene was initially identified on the basis of primary sequence homology and structural similarity. Subsequent characterization has confirmed its predicted activity as a protease inhibitor. Alterations of the hemostatic system can result from such causes as neoplasia and trauma. Such alterations result in an increased incidence of thrombotic disorders such as venous thrombosis. pulmonary embolism, atrial fibrillation, cerebral thrombosis, and hemophilia. Thus, there is a need for identification and characterization of polypeptides that function as inhibitors of the coagulation pathway which can play a role in detecting, preventing, ameliorating or correcting such disorders.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding at least a portion of the TFPI-3 polypeptide having the complete amino acid sequence shown in SEQ ID NO:2 or the complete amino acid sequence encoded by the cDNA clone deposited as plasmid DNA as ATCC Deposit Number 97797 on Nov. 20, 1996. The nucleotide sequence determined by sequencing the deposited TFPI-3 clone, which is shown in FIGS, 1A and 1B (SEQ ID NO:1), contains an open reading frame encoding a complete polypeptide of 252 amino acid residues, including an initiation codon encoding an N-terminal methionine at nucleotide positions 361 to 363 and a predicted molecular weight of about 28.2 kDa.
The TFPI-3 protein of the present invention shares sequence homology with the translation product of the human mRNA for Tissue Factor Pathway Inhibitor (TFPI), TFPI-2 and aprotinin, including the following conserved domains: (a) a first predicted Kunitz-type domain of about 51 amino acids; and (b) a second predicted Kunitz-type domain also of about 51 amino acids, both of which are thought to be important in regulating blood coagulation, and are underscored with stars in FIGS. 1A and 1B. The homology between human TFPI, TFPI-2, aprotinin and TFPI-3, shown in FIG. 2, indicates that TFPI-3 is also a protease and may be involved in regulating blood coagulation. Protease inhibition assays described herein confirm the ability of TFPI-3 polypeptides to inhibit protease activity.
The encoded polypeptide has a predicted leader sequence of 27 amino acids underlined in FIGS. 1A and 1B; and the amino acid sequence of the predicted mature TFPI-3 protein is also shown in FIGS. 1A and 1B, and as amino acid residues 1-225 (SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding a polypeptide comprising the predicted second Kunitz-type domain of the TFPI-3 polypeptide having the amino acid sequence at positions 106 to 156 in SEQ ID NO:2 or as encoded by the cDNA clone contained in ATCC Deposit No. 97797; (b) a nucleotide sequence encoding a polypeptide comprising the consensus Kunitz-type domain having the amino acid sequence shown as SEQ ID NO:28; and (c) a nucleotide sequence complementary to any of the nucleotide sequences in (a) or (b) above; wherein said nucleic acid sequence in (a) or (b) does not encode a polypeptide comprising a sequence shown as SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical. and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b) or (c), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b) or (c), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a TFPI-3 polypeptide having an amino acid sequence in (a) or (b), above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TFPI-3 polypeptides or peptides by recombinant techniques.
The invention further provides an isolated TFPI-3 polypeptide comprising an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the polypeptide comprising the predicted second Kunitz-type domain of the TFPI-3 polypeptide having the amino acid sequence at positions 106 to 156 in SEQ ID NO:2 or as encoded by the cDNA clone contained in ATCC Deposit No. 97797; or (b) the amino acid sequence of the polypeptide comprising the consensus Kunitz-type domain having the amino acid sequence of SEQ ID NO:28; wherein said nucleic acid sequence in (a) or (b) does not encode a polypeptide comprising a sequence shown as SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31.
The polypeptides of the present invention also include polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those described in (a) or (b) above, as well as polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity, to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which comprises the amino acid sequence of an epitope-bearing portion of a TFPI-3 polypeptide having an amino acid sequence described in (a) or (b), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a TFPI-3 polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention.
In another embodiment, the invention provides an isolated antibody that binds specifically to a TFPI-3 polypeptide having an amino acid sequence described in (a) or (b) above. The invention further provides methods for isolating antibodies that bind specifically to a TFPI-3 polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
The invention also provides for pharmaceutical compositions comprising TFPI-3 polypeptides, particularly human TFPI-3 polypeptides, which may be employed, for instance, in inhibiting intravascular clotting and preventing the formation of fribrin clots both in vitro and in vivo, for anticoagulant therapy in prophylaxis of venous thrombosis and as treatment for preventing its extension, as well as to provide low-dose regiment for prevention of postoperative deep venous thrombosis and pulmonary embolism, for the prophlaxis and treatment of pulmonary embolism and atrial fibrillation with embolism, to prevent clotting in arterial and heart surgery as well as for prevention of cerebral thrombosis in evolving stroke, for treating coronary occlusion with acute myocardial infarction and in the prophylaxis and treatment of peripheral arterial emoblism, for the treatment of sepsis, inflamatory diseases and transplant rejection, in the treatment of hyperfibrinolytic hemorrhage and traumatic hemorrhagic shock as well as in diseases connected with excessive release of pancreatic elastase (pancreatitis), serum elastase (artherosclerosis), leukocyte elastase in acute and chronic inflammation with damage to connective tissue, in damage to vessel walls, in necrotic diseases, and in degeneration of lung tissue. Methods of treating individuals in need of TFPI-3 polypeptides are also provided.
The invention further provides compositions comprising a TFPI-3 polynucleotide or a TFPI-3 polypeptide for administration to cells in vitro, to cells ex vivo and to cells in vivo, or to a multicellular organism. In certain particularly preferred embodiments of this aspect of the invention, the compositions comprise a TFPI-3 polynucleotide for expression of a TFPI-3 polypeptide in a host organism for treatment of disease. Particularly preferred in this regard is expression in a human patient for treatment of a dysfunction associated with aberrant endogenous activity of a TFPI-3.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a biological activity of the TFPI-3 polypeptide, which involves contacting a protease which is inhibited by TFPI-3 polypeptide with the candidate compound in the presence of a TFPI-3 polypeptide and a substrate cleavable by the selected protease, assaying the inhibitory activity of the protease activity of the protease in the presence of the candidate compound and of TFPI-3 polypeptide, and comparing the protease activity to a standard level of activity, the standard being assayed when contact is made between the protease and substrate in the presence of the TFPI-3 polypeptide, and in the absence of the candidate compound. In this assay, an increase in inhibitory activity over the standard indicates that the candidate compound is an agonist of TFPI-3 activity and a decrease in inhibitory activity compared to the standard indicates that the compound is an antagonist of TFPI-3 activity.
It has been discovered that TFPI-3 is expressed at differing levels in some tissues. Therefore, nucleic acids of the invention are useful as hybridization probes for differential identification of the tissue(s) or cell type(s) present in a biological sample. Similarly, polypeptides and antibodies directed to those polypeptides are useful to provide immunological probes for differential identification of the tissue(s) or cell type(s). In addition, for a number of disorders of the above tissues or cells, particularly of the hemostatic system, significantly higher or lower levels of TFPI-3 gene expression may be detected in certain tissues (e.g., cancerous and wounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from an individual having such a disorder, relative to a xe2x80x9cstandardxe2x80x9d TFPI-3 gene expression level, i.e., the TFPI-3 expression level in healthy tissue from an individual not having the hemostatic system disorder. Thus, the invention provides a diagnostic method useful during diagnosis of such a disorder, which involves: (a) assaying TFPI-3 gene expression level in cells or body fluid of an individual; (b) comparing the TFPI-3 gene expression level with a standard TFPI-3 gene expression level, whereby an increase or decrease in the assayed TFPI-3 gene expression level compared to the standard expression level is indicative of disorder in the hemostatic.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of TFPI-3 activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated TFPI-3 polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of TFPI-3 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an TFPI-3 antagonist. Preferred antagonists for use in the present invention are TFPI-3-specific antibodies and TFPI-3 proteins having an amino acid residue other than arginine at the P1 residue of either the first or second Kunitz-type domain, positions 21 and 116 of SEQ ID NO:2, respectively.